Introduction

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The liver plays a significant role in the detoxification of drugs and other xenobiotics as does the kidney. The uptake of organic anions from the circulating blood into the liver is the first step in any hepatic elimination. The hepatic uptake of organic anions has been characterized in vivo, in situ, and in vitro by many researchers and the process is generally considered to be mediated by transporters (Muller and Jansen, 1997; Kullak-Ublick, 1999; Meijer et al., 1999; Suzuki and Sugiyama, 1999). There are sodium-dependent and sodium-independent uptake mechanisms for the hepatic uptake of organic anions. According to the results of kinetic analyses, these two transport mechanisms accept a variety of structurally unrelated organic anions (Muller and Jansen, 1997; Kullak-Ublick, 1999; Meijer et al., 1999; Suzuki and Sugiyama, 1999). The transporter(s) responsible for the sodium-dependent uptake of organic anions remains to be isolated. Rat organic anion-transporting polypeptides (rOatp1, rOatp2, and rOatp4) have been identified as candidates for the sodium-independent uptake mechanism in the rat liver (Muller and Jansen, 1997; Kullak-Ublick, 1999; Meijer et al., 1999; Suzuki and Sugiyama, 1999; Cattori et al., 2000). OATPs accept amphipathic organic anions, such as bromosulfophthalein, bile acids, and glucuronide and sulfate conjugates of steroids (Muller and Jansen, 1997; Stieger and Meier, 1998; Kullak-Ublick, 1999; Suzuki and Sugiyama, 1999). They are considered to play key roles in the hepatic uptake of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (Hsiang et al., 1999; Tokui et al., 1999), a cyclic peptide BQ-123 (Reichel et al., 1999), a thrombin inhibitor CRC-220 (Eckhardt et al., 1996), and two angiotensin-converting enzyme inhibitors enalapril (Pang et al., 1998) and temocaprilat (Ishizuka et al., 1998). However, no significant uptake of ibuprofen and IDM was observed in rOatp1-transfected COS-7 cells (Kouzuki et al., 1999, 2000). In addition, the transporter responsible for the hepatic uptake of bumetanide has been suggested not to be rOatp1 (Horz et al., 1996; Petzinger et al., 1996). Additional uptake systems for organic anions are expected to be involved in the hepatic uptake of organic anions.

Rat organic anion transporter 1 (rOat1) has been cloned from rat kidney (Sekine et al., 1997; Sweet et al., 1997). It is abundantly expressed in the kidney and localized to the basolateral membrane of the proximal tubules (Tojo et al., 1999). Functional analyses using the Xenopus laevis oocyte expression system have shown that rOat1 mediates the transport of various kinds of organic anions such as p-aminohippurate (PAH) (Sekine et al., 1997), NSAIDs (Tsuda et al., 1999), -lactam antibiotics (Jariyawat et al., 1999), dicarboxylates, cyclic nucleotides, folate, antiviral nucleoside analogs (Wada et al., 2000), and thiazide diuretics (Uwai et al., 2000). In contrast, rOat2 is abundantly expressed in the liver and, to a much lesser extent, in the kidney, and localized to the basolateral membrane of the liver (Simonson et al., 1994; Sekine et al., 1998). Sekine et al. (1998) characterized rOat2 using X. laevis expression system and demonstrated that rOat2 transports several organic anions, such as salicylate (K_m of 89 µM), -ketoglutarate (K_m of 18 µM), methotrexate (MTX), prostaglandin E₂, and PAH. Therefore, it is possible that rOat2 is also involved in the hepatic uptake of these organic anions.

In this study, rOat2-expressing mammalian cells were obtained using LLC-PK1 cells and further experiments were carried out to investigate substrate specificity.

	Experimental Procedures

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Materials. [¹⁴C]Salicylate (55.5 mCi/mmol), [¹⁴C]indomethacin (IDM, 20 mCi/mmol), [¹⁴C]-ketoglutarate (-KG, 273.1 mCi/mmol), and [³H]PAH (4.08 Ci/mmol) were obtained from PerkinElmer Life Science Products (Boston, MA); [³H]2',3'-dideoxycytidine (ddC, 50 Ci/mmol) and [³H]3'-azido-3'-deoxythymidine (AZT, 11.7 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA), and [³H] prostaglandin E₂ (165 Ci/mmol) was obtained from Amersham Pharmacia Biotech UK (Buckinghamshire, England). All cell culture media and reagents were obtained from Invitrogen (Gaithersburg, MD), except fetal bovine serum (from Cancera, ON, Canada). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.

Stable Transfection of LLC-PK1 Cells with rOat2. LLC-PK1 cells were cultured in medium containing M199 supplemented with 10% fetal bovine serum. The complete coding and noncoding region of rOat2 was cut out from the original plasmid using XbaI and KpnI. This region was inserted into pcDNA3.1(+) mammalian expression vector (Invitrogen, Carlsbad, CA). Transfection of this construct was carried out using LipofectAMINE according to the manufacturer's protocol. The cells were maintained in a selection medium containing G418 (600 µg/ml) to select gene-transfected cells. Among the G418-resistant clones, the stable transfectants expressing rOat2 were selected by Northern blot analysis. One clone, which exhibited the highest transport activity for salicylate, was maintained in the presence of G418 (400 µg/ml) and used in all subsequent experiments.

Uptake Studies in LLC-PK1 Cells. Expression of rOat2 was induced by incubating cells for 24 h in the presence of sodium butyrate (5 mM) before starting the transport experiments as described previously (Eckhardt et al., 1999). To obtain the kinetic parameters, the linear range of uptake was determined for each substrate. The uptake was measured at a time point within this linear range and net uptake values used for the calculation were obtained by subtracting the uptake values into vector-transfected LLC-PK1 cells from those into rOat2-expressing LLC-PK1 cells. All transport assays were performed in Krebs-Henseleit buffer (142 mM NaCl, 23.8 mM Na₂CO₃, 4.83 mM KCl, 0.96 mM KH₂PO₄, 1.20 mM MgSO₄, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl₂ adjusted to pH 7.4). The composition of the choline buffer was the same as that of the Krebs-Henseleit buffer, except that NaCl and NaHCO₃ were replaced by choline chloride and choline bicarbonate, respectively.

Kinetic Analyses. Kinetic parameters were obtained using the following equation:
where v is the uptake rate of the substrate (pmol/min/mg of protein), S is the substrate concentration in the medium (µM), K_m is the Michaelis-Menten constant (µM) and V_max is the maximum uptake rate (pmol/min/mg of protein). To obtain the kinetic parameters, the equation was fitted to the rOat2-mediated uptake velocity, which was calculated by subtracting the uptake value of substrate into vector-transfected LLC-PK1 cells from that into rOat2-expressing cells. Fitting was performed using a MULTI program. The input data were weighted as the reciprocal of the observed values, and the Damping Gauss Newton Method algorithm was used for fitting. Inhibition of the rOat2-mediated uptake of [¹⁴C]salicylate (5 µM) was examined at 2 min, which is within the linear range of uptake. Inhibition constants (K_i) of several compounds were calculated assuming competitive inhibition.

Northern Blot Analysis. A 2-µg sample of mRNA, extracted from vector-transfected LLC-PK1 cells and rOat2-transfected cells using ISOGEN (Nippon Gene, Tokyo, Japan) and Oligotex dT30 super (Takara, Tokyo, Japan) according to manufacturer's protocol, was electrophoresed on 1% agarose/formaldehyde gel and transferred onto a nitrocellulose filter. The filter was hybridized in hybridization solution at 42°C with a full-length cDNA of rOat2 randomly labeled with [³²P]dCTP. The filter was then washed with 0.1% standard saline citrate/0.1% SDS at 55°C.

Antiserum and Western Blot Analysis. rOat2 antiserum was raised in rabbits against a synthetic peptide consisting of the 16 carboxyl-terminal amino acids of rOat2 coupled to keyhole limpet hemocyanine at its carboxyl terminus via an additional cysteine. Membrane fractions were prepared as previously described (Ogawa et al., 2000) and diluted with sample buffer without reducing agent and denatured at 95°C for 2 min before separation on 3.75% stacking and 10% resolving SDS polyacrylamide gels. Proteins were transferred electrophoretically onto polyvinylidene difluoride membrane (Pall Biosupport, East Hills, NY) using a blotter (Trans-blot; Bio-Rad, Richmond, CA) at 15 V for 1 h. The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% bovine serum albumin for 1 h at room temperature. After being washed with TBS-T (3 × 10 min), the membrane was incubated with anti-rOat2 serum (dilution 1:500). The membrane was allowed to bind ¹²⁵I-labeled sheep anti-rabbit IgG antibody (Amersham Pharmacia Biotech) diluted 1:200 in TBS-T containing 5% bovine serum albumin for 1 h at room temperature and were washed with TBS-T (3 × 5 min). Then the membrane was exposed to Fuji imaging plates (Fuji Photo Film, Kanagawa, Japan) for 3 h at room temperature, and analyzed with an imaging analyzer (BAS 2000; Fuji Photo Film).

	Results

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Expression of rOat2 in LLC-PK1 Cells. The expression of rOat2 in transfected cells was confirmed by Northern and Western blot analyses. As shown in Fig. 1a, the rOat2 transcript was found at approximately 2.6 and 2.2 kb in the rOat2-expressed LLC-PK1 cells and the liver (lanes 2 and 3). Following Western blot analysis, rOat2 protein was detected at about 63 and 52 kDa in the rOat2-expressed LLC-PK1 cells and in rat liver, respectively (Fig. 1b, lanes 1 and lane 3). The molecular mass of rOat2 in the liver was slightly lower than that in rOat2-expressed LLC-PK1 cells. The band was abolished when the preabsorbed antiserum for rOat2 was used (Fig. 1, lanes 4-6), suggesting that the positive band were specific for the antigen peptide. No expression of porcine OAT2 was observed in vector-transfected LLC-PK1 cells (Fig. 1, a and b, lane 2).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Expression of rOat2. Expression of rOat2 in rOat2-expressed LLC-PK1 cells and liver was examined by Northern (a) and Western (b) blot analyses. a, mRNA (2 µg/lane) prepared from rOat2-expressed LLC-PK1 cells (lane 1), vector-transfected LLC-PK1 cells (lane 2), and liver (lane 3) were electrophoresed on 1% agarose/formaldehyde gel. They were transferred to a nitrocellulose filter. The membrane was probed with 32P-labeled cDNA of rOat2. b, crude membrane prepared from rOat2-expressed and vector-transfected LLC-PK1 cells and the liver were used in Western blot analysis. The membrane was incubated with anti-rOat2 serum (lanes 1-3) or with preabsorbed anti-rOat2 serum (lanes 4-6). Lanes 1, 2, 4, and 5 were loaded with 10 µg of crude membrane from rOat2-expressed and vector transfected LLC-PK1 cells, and lanes 3 and 6 were loaded with 25 µg of crude liver membrane.

Characterization of rOat2-Mediated Salicylate Uptake. The time profile of the uptake of [¹⁴C]salicylate via rOat2 is shown in Fig. 2a. The intracellular accumulation of salicylate was significantly greater in rOat2-expressed LLC-PK1 cells than that in vector-transfected LLC-PK1 cells. Replacement of sodium by choline in the transport buffer has no effect on the transport of salicylate via rOat2 (Fig. 2b). The uptake was saturated at higher substrate concentrations (Fig. 2c). The K_m and V_max values were found to be 81.6 µM and 1040 pmol/min/mg of protein, respectively (Fig. 2c; Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Time profile (a), sodium dependence (b), and Eadie-Hofstee plot (c) of the uptake of [14C]salicylate into rOat2-expressed LLC-PK1 cells. The uptake of [14C]salicylate (5 µM) into rOat2-expressed LLC-PK1 cells was measured in Krebs-Henseleit buffer at 37°C. Open and closed circles represent the uptake of salicylate into vector-transfected and rOat2-LLC-PK1 cells in a and c, respectively. Open and closed columns in b represent the uptake of salicylate into vector- and rOat2-transfected cells, respectively. The uptake clearance was obtained from the net uptake value divided by the substrate concentration in the medium. Each data point represents the mean and S.E. (n = 3).

Substrate Specificity of rOat2-Meditated Transport. Transfection of rOat2 resulted in an increase in the intracellular accumulation of prostaglandin E₂, IDM, ddC, and AZT (Fig. 3). The kinetic parameters (K_m and V_max values) are listed in Table 1. The uptake of PAH into rOat2-expressed LLC-PK1 cells was slightly higher than that into vector-transfected LLC-PK1 cells (6.25 ± 0.46 and 4.32 ± 0.06 µl/30 min/mg of protein, respectively) (p < 0.05). No significant uptake of -KG, MTX, and glibenclamide into rOat2-expressed LLC-PK1 cells was observed. The uptake into vector and rOat2-expressed LLC-PK1 cells was 11.0 ± 2.1 and 11.6 ± 1.1 µl/30 min/mg of protein (for -KG), 9.67 ± 0.39 µl/30 min/mg of protein, and 11.3 ± 0.5 µl/30 min/mg of protein (for MTX), 9.89 ± 0.52 µl/15 min/mg of protein, and 10.2 ± 0.30 µl/15 min/mg of protein (for glibenclamide), respectively.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Time profiles of the uptake of IDM, ddC, AZT, and PGE2. The uptake of IDM (0.14 µM), ddC (12.5 nM), AZT (25 nM), and PGE2 (6 µM) into rOat2-expressed and vector-transfected LLC-PK1 cells was measured. Open and closed circles represent the uptake of salicylate into vector-transfected and rOat2-expressed cells, respectively. Each data point represents the mean ± S.E. (n = 3).

Inhibition Study. Indocyanine green, ketoprofen, and glibenclamide exhibited the strongest potency with K_i values of 1.15, 1.84, and 12.3 µM, respectively. Diclofenac and benzoate were moderate inhibitors (K_i of 49.3 and 86.9 µM, respectively) while verapamil, tolbutamide, and ibuprofen were the weakest inhibitors (K_i of 140-180 µM) (Fig. 4). The inhibition constants (K_i values) of these drugs are summarized in Table 2. The inhibitory effects of -KG, phenytoin, cimetidine, digoxin, propionate, MTX, PAH, and benzylpenicillin on the uptake of salicylate via rOat2 were either very weak or completely absent (Fig. 5).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Inhibitory potencies of eight compounds on rOat2-mediated uptake of [14C]salicylate. The uptake of [14C]salicylate (5 µM) was examined in the presence of inhibitors at the concentrations (conc) indicated. The values are expressed as a percentage of rOat2-mediated uptake of [14C]salicylate in the absence of any inhibitor. Solid lines represent the fitted line obtained by nonlinear regression analysis. Each data point represents the mean ± S.E. (n = 3).

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of unlabeled compounds on rOat2-mediated of [14C]salicylate. The uptake of [14C]salicylate (5 µM) was measured in the presence of inhibitors at 1 mM, except for digoxin and rifampicin (0.2 mM). The values are expressed as a percentage of the Oat2-mediated uptake of salicylate in the absence of any inhibitor. Each data point represents the mean ± S.E (n = 3).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In the present study, rOat2-expressed LLC-PK1 cells were constructed and the substrate specificity of rOat2 was characterized.

The expression of rOat2 cDNA was studied in the transfected LLC-PK1 cells (Fig. 1). Northern blot analysis indicated that the length of the transcript (approximately 2.6 kb) was slightly longer than that observed in the liver (2.2 kb). This difference was partially accounted for by the fact that the transcript in rOat2-expressed LLC-PK1 cells contains small fragments derived from the vector (pcDNA3.1; from KpnI site to a polyadenylation signal of the vector) at the 3' end of full-length cDNA of rOat2. Western blot analysis indicated that the molecular mass of rOat2 expressed in LLC-PK1 cells (approximately 60 kDa) agreed with the previously reported value in the liver (Simonson et al., 1994). The molecular mass of rOat2 in the liver was slightly lower than that in rOat2-expressed LLC-PK1 cells and the previously reported value for some unknown reason. Further studies are required to reveal the reason.

The uptake of salicylate into rOat2-expressed LLC-PK1 cells was significantly higher than that into vector-transfected LLC-PK1 cells and independent of the presence of sodium in the medium (Fig. 2). The K_m and V_max values were 81 µM and 1410 pmol/min/mg of protein, respectively (Table 1; Fig. 2). These results are consistent with a previous observation (K_m = 88.8 µM in rOat2-expressed oocytes; Sekine et al., 1998). However, no specific uptake of -KG was observed in rOat2-expressed LLC-PK1 cells, although -KG was shown to be a good substrate for rOat2 using rOat2-expressed oocytes (Sekine et al., 1998). -KG does not affect the uptake of salicylate into rOat2-expressed LLC-PK1 cells even at concentration (1 mM) much higher than the K_m value determined using rOat2-expressed oocytes (18 µM). Therefore, reduced transport of -KG in rOat2-expressed LLC-PK1 cells is due to the lack of an interaction between -KG and rOat2 in rOat2-expressed LLC-PK1 cells. The reason for this is unknown but it is possible that differences in microenvironment, such as lipid composition, may have affected the substrate specificity of rOat2. Further studies are required to investigate which expression system most accurately reflects the in vivo situation.

IDM and nucleoside derivatives, such as AZT and ddC, were identified for the first time as rOat2 substrates in the present study. IDM is the substrate with the highest affinity for rOat2 (K_m = 0.4 µM; Table 1). Based on the inhibition study, indocyanine green, ketoprofen, glibenclamide, benzoate, and diclofenac are considered as possible substrates of rOat2 (Fig. 4). However, as far as glibenclamide is concerned, there was no significant difference in the uptake between rOat2-expressed and vector-transfected LLC-PK1 cells.

As demonstrated previously, rOat1 also transports IDM, salicylate, AZT, and ddC (Apiwattanakul et al., 1999; Wada et al., 2000). The K_m value of AZT for rOat2 was comparable with that for rOat1 (26 and 68 µM, respectively). ddC reduced rOat1-mediated PAH uptake to 70% of the control value even at 1 mM (Wada et al., 2000). ddC is a low-affinity substrate of both rOat1 and rOat2, which have similar affinity for nucleoside analogs. A clear difference was observed in NSAIDs and PAH transport. The K_m values of IDM and salicylate for rOat1 were 30- and 5-fold greater than those for rOat2 (10 and 0.4 µM, and 340 and 81 µM, respectively) and IDM does not inhibit the uptake of estrone sulfate in rOat3-expressed oocytes (Kusuhara et al., 1999). These NSAIDs have higher affinities for rOat2 than for rOat1 and rOat3. The affinity of PAH for rOat2 is much lower than those for rOat1 (Sekine et al., 1997) and rOat3 (Kusuhara et al., 1999) (14 and 65 µM determined using oocytes, respectively), since it does not affect the uptake of salicylate via rOat2 even at 1 mM (Fig. 5). In addition, the transport activity of PAH by rOat2 was quite low, if present at all. These results are consistent with the observation that the distribution volume of PAH in the liver is very close to the capillary space after intravenous administration (M. Hasegawa, unpublished data).

The hepatic uptake process of IDM was characterized using primary cultured rat hepatocytes (Kouzuki et al., 2000). The uptake of IDM into primary cultured hepatocytes exhibited some sodium dependence and the sodium-independent uptake accounts for 50% of the total uptake (Kouzuki et al., 2000). No significant uptake of IDM was observed in rat sodium taurocholate-cotransporting polypeptide- and rOatp1-expressed COS-7 cells (Kouzuki et al., 2000). The IC₅₀ values of typical substrates of OATPs such as taurocholate, pravastatin, dibromosulfophthalein, and estradiol 17 glucuronide for the uptake of IDM into rat primary cultured hepatocytes were larger than their own K_m values (Kouzuki et al., 2000). These results suggest that another organic anion transporter(s) is involved. The K_m value of IDM was determined to be 12 µM in hepatocytes (Kouzuki et al., 2000), which is much higher than that for rOat2 (K_m = 0.37 µM; Table 1). Since the lowest substrate concentration used in the hepatic uptake study was 1 µM, it is possible that the component, which rOat2 accounts for, was saturated even at the lowest concentration used in that experiment. Further studies are required to examine the contribution of rOat2 to the total hepatic uptake of IDM. The uptake of AZT into isolated hepatocytes was linear up to 250 µM and even at 4°C, 80% of the total uptake remained (Bezek et al., 1994), suggesting transporters make only a minor contribution of transporters to the total hepatic uptake of AZT, if at all, although AZT is a good substrate of rOat2. The hepatic uptake of ddC has not yet been characterized and an involvement of transporters, including rOat2 in its hepatic uptake process remains to be examined. As observed in the case of AZT, it is necessary to evaluate the contribution of a transporter to the total membrane transport process as well as to demonstrate that a drug is a substrate of the transporter. Inhibitors selective for each transporter are useful for this purpose. By examining their inhibitory effect, it is possible to evaluate the contribution of each transporter. However, little information is available about the selectivity of inhibitors for organic anion transporters. IDM, indocyanine green, ketoprofen, and glibenclamide are good inhibitors for rOat2 (Table 2; Fig. 5). It is necessary to confirm their selectivity for other organic anion transporters expressed in the liver (OATPs and rOat3) to evaluate the contribution of rOat2 to the total hepatic uptake of its ligands.

In conclusion, we have described the multispecificity of transport via rOat2 and identified a number of novel substrates and potent inhibitors. So, rOat2 may be involved in the hepatic uptake of organic anions with different structures, such as NSAIDs and nucleoside derivatives.